Cell wall polymers of Enterococcus faecalis and uses thereof

Cross reference to related applications

This application is a National Stage Application of International Application Number PCT/EP2013/054274, filed Mar. 4, 2013; which claims priority to European Patent Application No. 12162640.2, filed Mar. 30, 2012; which are incorporated herein by reference in their entirety.

The present invention relates to enterococcal cell wall polymers and their uses in the prevention and therapy of bacterial infection.

Background of the invention

Enterococcus faecalis is an important nosocomial pathogen and a frequent cause of infection in critically ill patients (Vincent J L, et al. International study of the prevalence and outcomes of infection in intensive care units. JAMA. 2009 Dec. 2; 302(21): 2323-9). Underlying malignancy, neutropenia, antineoplastic chemotherapy and immunosuppressive medication are well-characterized risk factors for invasive infections with enterococci (Ghanem G, et al. Outcomes for and risk factors associated with vancomycin-resistant Enterococcus faecalis and vancomycin-resistant Enterococcus faecium bacteremia in cancer patients. Infect Control Hosp Epidemiol. 2007 September; 28(9): 1054-9; DiazGranados C A, Jernigan J A. Impact of vancomycin resistance on mortality among patients with neutropenia and enterococcal bloodstream infection. J Infect Dis. 2005 Feb. 15; 191(4):588-95; Peel T, et al. Differing risk factors for vancomycin-resistant and vancomycin-sensitive enterococcal bacteraemia. Clin Microbiol Infect. 2012 April; 18(4):388-94. Epub 2011 Aug. 16) and the clinical outcome of invasive enterococcal infections in this patient population is frequently poor (Theilacker C, Jonas D, Huebner J, Bertz H, Kern W. Outcomes of Invasive Infection due to Vancomycin-Resistant Enterococcus faecium during a Recent Outbreak. Infection. 2009 December; 37(6):540-3). Risk factors that may contribute to the increased susceptibility of high-risk groups to enterococcal infection include high-density colonization of the gastrointestinal tract (Ubeda C, et al. Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans. J Clin Invest. 2010 Dec. 1; 120(12): 4332-41), damage to the gastrointestinal mucosal barrier, indwelling devices such as central venous catheters, and immune dysregulation (Peel T, et al. Differing risk factors for vancomycin-resistant and vancomycin-sensitive enterococcal bacteraemia. Clin Microbiol Infect. 2012 April; 18(4):388-94. Epub 2011 Aug. 16).

The complement system is an important first line of defense against invasive infections and plays a critical role in the immuno-compromised host with defective adaptive immunity. It comprises more than 30 proteins, which are detectable in human serum, on cell surfaces and in tissue fluids. The classical-, alternative- and lectin pathway all converge in the cleavage of C3 to form C3b, the key effector molecule of the complement system. Complement receptor 3 of neutrophils binds C3b deposited on the bacterial envelope leading to phagocytosis and killing of the ingested bacterium. Studies in C3-depleted mice have demonstrated that C3 is also critical for opsonphagocytosis and clearance of enterococci from infected organs (Leendertse M, et al. The complement system facilitates clearance of Enterococcus faecium during murine peritonitis. J Infect Dis. 2010 Feb. 15; 201(4): 544-52). In addition, multiple epidemiologic studies have shown that a deficiency of mannose-binding lectin—the best described factor of the lectin pathway—predisposes to severe infection and bacteremia in neonates, neutropenic patients and patients after allogeneic stem cell or solid organ transplantation (Vekemans M, et al. Low mannose-binding lectin concentration is associated with severe infection in patients with hematological cancer who are undergoing chemotherapy. Clin Infect Dis. 2007 Jun. 15; 44(12): 1593-601; Schlapbach L J, et al. Differential role of the lectin pathway of complement activation in susceptibility to neonatal sepsis. Clin Infect Dis. 2010 Jul. 15; 51(2): 153-62; Worthley D L, et al. Donor mannose-binding lectin deficiency increases the likelihood of clinically significant infection after liver transplantation. Clin Infect Dis. 2009 Feb. 15; 48(4):410-7; Mullighan C G, et al. Mannose-binding lectin status is associated with risk of major infection following myeloablative sibling allogeneic hematopoietic stem cell transplantation. Blood. 2008 Sep. 1; 112(5): 2120-8).

In the course of evolution, Gram-positive bacteria have developed numerous strategies to escape recognition and targeting by the complement system. The flexibility of pathogens to circumvent binding of complement factors is illustrated impressively by Staphylococcus aureus (Serruto D, Rappuoli R, Scarselli M, Gros P, van Strijp J A. Molecular mechanisms of complement evasion: learning from staphylococci and meningococci. Nat Rev Microbiol. 2010 June; 8(6): 393-9). In contrast, little is known about the interaction of E. faecalis and the complement system.

Toivanen P et al.: “Complement-fixing antibodies to adenovirus in rabbits and guinea-pigs treated with 6-mercaptopurine or epsilon-aminocaproic acid”, 1965, Acta Pathologica et Microbiologica Scandinavica 1965, Vol. 63, pages 221-227 and T Behr, et al. “The structure of pneumococcal lipoteichoic acid. Improved preparation, chemical and mass spectrometric studies” FEBS J 207(3):1063-75

describe compounds which comprise a group consisting of β-D-GalpNac-ribitol structures. However, structures wherein R1 is selected from β-D-Glcp or α-L-Rhap and R2 is selected from β-D-Glcp or H are not disclosed.

WO 2011/088843 describes enterococcal cell wall components and their uses in the prevention and therapy of bacterial infection.

In order to provide more efficient strategies to effectively treat and/or prevent infection in vertebrates caused, at least in part, by enterococci, new antigenic bacterial targets are needed which could be used in new and improved vaccination strategies, as well as in the development and production of respective vaccines.

Glycolipids, teichoic acids (TA) and wall teichoic acids (WTA) could serve as potential targets for new drugs for the treatment of Gram-positive bacterial infection. Although cell wall polysaccharide is a main component of the cell wall of Gram-positive bacteria as well, little is known about any potential antigens derived from this class of components.

As a part of the search for carbohydrate complement resistance factors and the development of alternative treatments such as glycoconjugate vaccines to combat enterococcal infections, the present invention fulfils these need by providing new capsular polysaccharides isolated from the cell wall of enterococci.

Thus, the objects of the present invention in a first aspect thereof are solved by an enterococcal cell wall component selected from the group consisting of β- D -GalpNAc-ribitol structures

##STR00001## wherein R.sub.1 is selected from β- D -Glcp or α- L -Rhap, and R.sub.2 is selected from H or α- D -Glcp, and derivatives thereof, and pharmaceutically acceptable salts thereof, wherein Rhap is 6-deoxy-mannopyranose (rhamnose); GalpNAc is 2-acetamido-2-deoxy-galactopyranose (N-acetyl-galactosamine); and Glcp is glucopyranose.

Preferably, R.sub.1 is selected from β- D -Glcp according to the following formula II

##str00002##

Preferably, R.sub.1 is selected from α- L -Rhap according to the following formula III

##str00003##

Preferably, R.sub.2 is selected from α- D -Glcp, according to the following formula IV

##STR00004## wherein the dashed lines in II to IV indicate the connection to the β- D -GalpNAc-ribitol structure as above.

Most preferred are the structures OS I and/or OS II as shown in FIG. 4 .

In the context of the present invention, the interaction of carbohydrate cell surface structures of E. faecalis with the human complement system was investigated. Previously, a library of 177 targeted insertion mutants of genes involved in putative surface or stress-response factors was constructed in E. faecalis strain V583, and the mutant library was screened for sensitivity to oponophagocytic killing and three mutants of genes putatively involved WTA biosynthesis were readily killed by complement and neutrophils in the absence of specific Ab (Rigottier-Gois L, et al. Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors. PLoS ONE. 2011 Dec. 15; 6(12): e29023). In contrast, wild type E. faecalis is resistant to killing by complement and neutrophils alone. The present invention is based on the mechanism of increased susceptibility to complement-mediated opsonphagocytosis and the characterization of structural differences of wild type and mutant cell wall accessory carbohydrate polymers.

Three insertional mutants of the wall teichoic acid (WTA) synthesis genes tagA, tagB and tagO in E. faecalis strain V583 were identified which exhibited an increased susceptibility to complement-mediated opsonophagocytosis by neutrophils. Further studies revealed a role of L-ficolin/mannose-binding lectin-associated serine protease (MASP) complexes in Ca.sup.2+-dependent, Ab-independent opsonophagocytosis of E. faecalis V583Δ1172. To understand the mechanism of lectin pathway activation by E. faecalis V583Δ1172, the inventors structurally characterized cell wall fragments of E. faecalis wild type and V583Δ1172 obtained by enzymatic digestion of peptidoglycan. Cell wall fragments of V583Δ1172 lacked the two oligosaccharides according to the present invention with the structure α- L -Rhap-(1.fwdarw.3)-β- D -GalpNAc-(1.fwdarw.1)-ribitol and α- D -Glcp-(1.fwdarw.4)-[β- D -Glcp-(1.fwdarw.3)-]β- D -GalpNAc-(1.fwdarw.1)-ribitol suggesting the absence of WTA in the mutant.

Thus, the enterococcal cell wall component (in the following also designated as “enterococcal antigen”) provides a new antigenic target for the development of more efficient strategies to effectively treat and/or prevent infection in vertebrates caused, at least in part, by enterococci, allow for improved vaccination strategies, and allow the development and production of respective vaccines, such as glycoconjugate vaccines.

According to the present invention, the term a “modified derivative” or “modified derivatives” shall include chemically or enzymatically modified enterococcal antigens according to the formula I as above, wherein said modified derivative maintains its function as an enterococcal antigenic determinant and/or to the same, or substantially the same, extent as the enterococcal antigen according to formula I. Preferably, said modified derivative exhibits a quantitatively increased immunological reaction, compared to a non-modified enterococcal antigen. Such increase of the immunological reaction can be detected with immunological assays known in the art.

Examples for modified derivatives are preferably compounds of formula I that are modified to include a linker group in order to be coupled or conjugated to other chemical entities. These linker groups are known in the state of the art, and usually are immunologically inactive, i.e. do not substantially interfere with the immunological properties of the enterococcal antigen. Other modifications include the addition of chemical moieties of the enterococcal antigen in order to carry a detectable label, such as chelating groups or enzymatic groups. Furthermore, peptide (e.g. His) or other “labels” or “tags” can be added in order to be able to purify and/or use the enterococcal antigen in diagnostic assays.

Finally, the enterococcal antigen can include chemical modifications, for example at the rings of the sugar components of the enterococcal antigen, wherein the antigen can be modified to replace an existing side group with either H, an unsubstituted, monosubstituted or polysubstituted C.sub.1-C.sub.18-alkyl, wherein said alkyl can be straight, branched or cyclic, alkenyl, an unsubstituted, monosubstituted or polysubstituted aryl or heteroaryl residue, an unsubstituted, monosubstituted or polysubstituted benzene group, an acyl group, such as formyl, acetyl, trichloroacetyl, fumaryl, maleyl, succinyl, benzoyl, or a branched or heteroatom or aryl substituted acyl group, an alkoxy substituent, such as —OMe, —OEt, —OnPr, -iPr, —OnBu, —OiBu, —OsecBu, —OtBu, whose alkyl group can be branched, straight or cyclic, an alkyl group bound via a sulphur atom such as —SMe, —SEt, or a sulfonyl group, such as —SO.sub.3H, —SO.sub.2Me, —SO.sub.2CF.sub.3, —SO.sub.2C.sub.6H.sub.4CH.sub.3 or SO.sub.2C.sub.6H.sub.4CH.sub.2Br, or a nitrogen substituent, such as NH.sub.2, NHR, —NRR′ (with R, R′=alkyl, alkenyl or aryl as above), NC or —NO.sub.2, or fluoro, chloro, bromo, iodine, —CN or a heterosubstituent. As mentioned above, these derivatives are preferably included in order to improve the solubility of the antigen, increase the immunological effect of said antigen (preferably quantitatively), and/or to allow the compound to be coupled to other moieties, e.g. in order to be coupled to a surface (such as a well or chip), and/or to be used in diagnostic assays.

Another aspect of the invention relates to a method for producing the enterococcal cell wall component according to the present invention, wherein said method comprises isolating said enterococcal cell wall component from a bacterial fraction, or comprising synthesizing said antigen, at least in part, through chemical synthesis. Isolation can include purifying said cell wall component from bacterial fractions to be substantially free of other bacterial components, but can also include the isolation as part of certain bacterial fractions, such as cell wall fractions including other parts of the cellular wall, as described herein.

Another aspect of the invention relates to an antibody, preferably a monoclonal antibody or antigenic fragment thereof, that specifically recognizes an enterococcal antigen according to the present invention. The term “antibody” shall include both monoclonal or polyclonal antibodies, recombinant antibodies or fragments thereof, such as Fab and the like, as well as human or humanized antibodies.

Another aspect of the invention then relates to a method for producing the antibody according to the present invention, comprising immunizing a mammal, preferably a rabbit, with an enterococcal cell wall component according to the present invention, or a with the pharmaceutical composition according to the present invention, and preferably the vaccine according to the present invention. Respective methods are known to the person of skill, and are disclosed in the state of the art.

Yet another aspect of the present invention then relates to a method for producing the antibody according to the present invention, comprising generating hybridoma cells producing said antibody as a monoclonal antibody, or comprising a recombinant production of said antibody in a host cell. Respective methods are known to the person of skill, and are disclosed in the state of the art

Still another important aspect of the present invention then relates to a strain of E. faecalis wherein insertional mutants in at least one of the WTA synthesis genes tagA, tagB and tagO are present, such as, for example, strain E. faecalis V583Δ1172. This strain can be used in assays and screening tests according to the present invention, e.g. for specific and/or effective antibodies as described herein, or for the screening and identification of specific compounds (such as small molecules) able to inhibit production of above-mentioned antigen.

Still another important aspect of the present invention then relates to the use of the enterococcal antigen according to the present invention as an antigen in the production of antibodies that are specific for said antigen.

Another aspect of the invention then relates to a pharmaceutical composition, comprising at least one enterococcal antigen according to the present invention and/or at least one antibody according to the present invention, together with a pharmaceutically acceptable carrier and/or excipient.

Particularly preferred is a pharmaceutical composition according to the present invention, wherein said composition comprises a cell wall component according to formula I, namely a respective cell wall polysaccharide.

Further preferred is a pharmaceutical composition according to the present invention, wherein said composition is formulated as a vaccine, in particular against infections caused by enterococci, in particular antibiotic-resistant enterococci, such as VRE strains, preferably of E. faecalis . Most preferred is a pharmaceutical composition according to the present invention, wherein said cell wall component according to formula I is present in a glycoconjugate vaccine.

The cell wall polysaccharide according to the present invention (either present as the antigen alone or in an extract or bacterium as described herein) is preferably used for an enterococci-vaccine, either for active or passive immunization.

Thus, according to the invention, there is provided a pharmaceutical composition, and in particular a vaccine, for the prevention of enterococcal infections in a vertebrate, said pharmaceutical composition comprising at least one new enterococcal antigen according to the present invention, optionally together with a pharmaceutically acceptable carrier, adjuvants and/or diluent.

Typically, the vaccine can comprise live or dead intact cells of at least one enterococcal strain, preferably of E. faecalis , comprising the enterococcal antigen of the invention. More typically, the vaccine comprises cell lysate from at least one of said enterococcal strains as comprising the enterococcal antigen or antigens. Even more typically, the vaccine comprises a crude enterococcal antigen mixture or purified enterococcal antigen or enterococcal antigens from at least one of said enterococcal strains, preferably E. faecalis . Still more typically, the vaccine comprises a fraction of the cell wall and associated proteins as enterococcal antigen of at least one of said enterococcal strains. The vaccine may also be comprised of a combination of one of the components. Most preferred is a glycoconjugate vaccine comprising an enterococcal antigen according to the present invention. Another aspect relates to a pharmaceutical composition or vaccine, wherein the enterococcal antigen as included has been produced, at least in part, through chemical synthesis. The methods for purifying the selected bacterial fractions containing enterococcal antigens are known to the person of skill, and are further described herein.

Typically, the vertebrate is a monogastric, herbivore or ruminant animal or human subject. Even more typically, the vertebrate is selected from the group consisting of human, non-human primate, murine, bovine, ovine, equine, porcine, caprine, leporine, avian, feline and canine. More typically, the vertebrate is selected from the group consisting of human, ovine, camelids, porcine, bovine, equine or canine.

The pharmaceutical composition can be formulated for administration via intramuscular, subcutaneous, topical or other parenteral route. In general, the microorganisms of the present invention are commensal in nature. Thus, oral administration is generally not an effective route of vaccination, and as a consequence, administration via an intramuscular, subcutaneous topical or other parenteral route is preferred. The vaccine may also include cytokines, such as: G-CSF, GM-CSF, interleukins or tumor necrosis factor alpha, used singly or in combination.

The pharmaceutical composition may also include an adjuvant. More typically, the adjuvant is selected from the group consisting of Freund's complete/incomplete adjuvant, montenide macrol adjuvant, phosphate buffered saline and mannan oil emulsions, saponins (QuiLA) dextran (dextran sulphate, DEAE-Dextran), aluminum compounds (Imject Alum), N-acetylglucosamiyl-N-acetylmuramyl- L -alanyl- D -isoglutamine (Gerbu adjuvant). More typically, the adjuvant is selected from the group as described in the Vaccine 1995, vol 13, p 1203; 1993 vol 11 p 293; and 1992 vol 10 p 427, the disclosures of which are incorporated herein by reference.

Yet another important aspect of the present invention then relates to an enterococcal cell wall component (enterococcal antigen) according to the present invention, the antibody according to the present invention, or the pharmaceutical composition according to the present invention for use in the treatment of diseases, such as bacterial infections, in particular enterococcal infection, such as nosocomial bacteremia infection, endocarditis, urinary tract infections, surgical wound infections, and foreign body infections.

Yet another important aspect of the present invention then relates to the use of the enterococcal cell wall component according to the present invention, the antibody according to the present invention, or the pharmaceutical composition according to the present invention (i.e. the enterococcal antigens) for the treatment against bacterial infections or for the preparation of a medicament against bacterial infections, in particular enterococcal infection, such as nosocomial bacteraemia infection, endocarditis, urinary tract infections, surgical wound infections, and foreign body infections, in particular caused by antibiotic-resistant gram-positive cocci, such as enterococci, VRE strains, or such as E. faecalis.

According to yet another preferred embodiment of the invention, there is provided a method for inducing an immune response against at least one enterococcal strain comprising the enterococcal antigen of the present invention in a vertebrate, said method comprising administering to said vertebrate an immunologically effective amount of the vaccine in accordance with the invention, or a pharmaceutical composition in accordance with the invention.

According to yet another preferred embodiment of the invention, there is provided a method for treating or preventing a bacterial infection in a vertebrate, comprising administering to said vertebrate a therapeutically effective amount of the enterococcal cell wall component according the present invention, the antibody according to the present invention, or the pharmaceutical composition according to the present invention.

Preferred is a method according to the present invention, wherein said bacterial infection is an enterococcal infection, such as nosocomial bacteraemia, endocarditis, a urinary tract infection, surgical wound infection, and foreign body infection, and is particular caused by antibiotic resistant enterococci, such as a VRE strain, and in particular E. faecalis.

To examine the role of WTA in the resistance to complement-mediated opsonophagocytosis, the inventors searched for E. faecalis genes with similarity to genes involved in the biosynthesis of WTA in Bacillus subtilis and Staphylococcus aureus using BLAST analysis. Compared to B. subtilis , the E. faecalis V583 chromosome carries only one teichoic acid glycerol (tag) operon, comprising TagB (EF_1172) and TagA (EF_1173). These two genes are followed by a gene of unknown function (EF_1174) and TagD (63% identity 80% similarity with TarA in B. subtilis W23, a glycerol-3-phosphate cytidylyltransferase; EF_1175). The most obvious homologue of the enzyme which catalyzes the first step of WTA biosynthesis, TagO, in V583 is protein EF_2198 (41% identity, 65% similarity with TagO in S. aureus COL). Previous work, however, has linked this gene also to the epa locus, which is the gene cluster involved in the biosynthesis of the enterococcal cell-wall polysaccharide, also called enterococcal polysaccharide antigen. Interestingly, a disruption mutant of EF_2198 in E. faecalis OG1RF still expressed the cell wall polysaccharide as assessed by agarose gel electrophoresis and reactivity to specific Abs (Teng F, Singh K V, Bourgogne A, Zeng J, Murray B E. Further characterization of the epa gene cluster and Epa polysaccharides of Enterococcus faecalis . Infect Immun. 2009 September; 77(9):3759-67).

In the opsonophagocytic assay, the inventors compared the killing of wild type bacteria and insertional mutants in tagO (EF_2198), tagA (EF_1173) and tagB (EF_1172) in the presence of complement and neutrophils alone or together with Abs specific to enterococcal cell surface antigens. In contrast to the wild type, all three mutants in WTA biosynthesis genes were highly susceptible to serum opsonophagocytic killing depleted of specific Ab. The higher killing strongly correlated with increased phagocytosis by neutrophils and a higher density of C3d bound to the bacterial surface of the mutant strain. Additional experiments excluded that the classical and alternative pathway were involved in the increased deposition of C3b on E. faecalis V583Δ1172 and provided evidence that complement deposition was due to activation of the lectin pathway.

In order to identify carbohydrate structures that may serve as ligands for the lectin pathway in E. faecalis V583Δ1172, the inventors investigated the structural differences in accessory cell wall polymers between mutant and wild type strain. After enzymatic cleavage of peptidoglycan, the inventors identified a PAS-positive band on SDS-PAGE analysis which has previously been identified as the enterococcal cell wall polysaccharide (Theilacker C, et al. Serodiversity of Opsonic Antibodies against Enterococcus faecalis glycans of the Cell Wall Revisited. PLoS ONE. 2011; 6(3):e17839).

In the deletion strain E. faecalis V583Δ1172, the characteristic band of the cell wall polysaccharide displayed a much slower migration pattern. In addition, the polysaccharide had lost its ability to bind to the cationic dye Stains All and also did not bind to Q-Sepharose, suggesting a loss of anionic charge. Using size-exclusion and anion-exchange chromatography, the inventors obtained purified cell wall fragments from the wild type E. faecalis V583 and the E. faecalis V583Δ1172 mutant. Material from the E. faecalis V583Δ1172 mutant contained less GalNAc, ribitol and phosphate, indicating a loss of WTA from the cell envelope of the mutant (Weidenmaier C, McLoughlin R M, Lee J C. The zwitterionic cell wall teichoic acid of Staphylococcus aureus provokes skin abscesses in mice by a novel CD4+ T-cell-dependent mechanism. PLoS ONE. 2010; 5(10):e13227; Neuhaus F C, Baddiley J. A continuum of anionic charge: structures and functions of D -alanyl-teichoic acids in gram-positive bacteria. Microbiol Mol Biol Rev. 2003 December; 67(4):686-723; Swoboda J G, Campbell J, Meredith T C, Walker S. Wall teichoic acid function, biosynthesis, and inhibition. Chembiochem. 2010 Jan. 4; 11(1):35-45).

A definitive elucidation of the structure of the purified native accessory cell wall polysaccharide by NMR spectroscopy was not possible because of the complex and heterogeneous nature of this carbohydrate in both strains. The inventors therefore depolymerized the cell wall fragments by dephosphorylation with HF. After fractionation by gel chromatography, the inventors obtained three distinct pools. The high molecular mass pool contained Rha, Glc, GlcN, and GalN, indicative of the enterococcal cell wall polysaccharide. Methylation analysis suggested a poly-Rha chain with terminal Glc and hexosamine residues. The medium-sized pool contained Rha, Glc, GlcN, GalN, and peptidoglycan fragments, as indicated by the presence of muramic acid and the amino acids Ala, Glu and Lys in a molar ratio of ˜4:1:1, consistent with the composition of E. faecalis peptidoglycan (Bouhss A, Josseaume N, Severin A, Tabei K, Hugonnet J E, Shlaes D, et al. Synthesis of the L -alanyl- L -alanine cross-bridge of Enterococcus faecalis peptidoglycan. J Biol Chem. 2002 Nov. 29; 277(48):45935-41).

Hence, the oligosaccharide likely represented the linkage unit of the cell wall polysaccharide to peptidoglycan. The small molecular mass pool consisted of GalN, ribitol, Glc, and Rha in a molar ratio of ˜3:2:1:1. After further purification steps, two oligosaccharides were detected which were amendable to NMR spectroscopy. The structural analysis revealed two ribitol-containing teichoic acid fragments, namely α- L -Rhap-(1.fwdarw.3)-β- D -GalpNAc-(1.fwdarw.1)-ribitol and α- D -Glcp-(1.fwdarw.4)-[β- D -Glcp-(1.fwdarw.3)-]β- D -GalpNAc-(1.fwdarw.1)-ribitol.

Of note, unsuccessful cleavage of a peptidoglycan bridge linking the streptococcal group B carbohydrate and capsular polysaccharide by mutanolysin has been described previously (Deng L, Kasper D L, Krick T P, Wessels M R. Characterization of the linkage between the type III capsular polysaccharide and the bacterial cell wall of group B Streptococcus . J Biol Chem. 2000 Mar. 17; 275(11):7497-504).

In summary, the inventors observed two major differences in the cell wall fragments of E. faecalis V583Δ1172 compared to the wild type: a) a higher Rha-content of the putative cell-wall polysaccharide and b) the loss of ribitol-containing teichoic acid in the mutant.

Studies in Gram-positive pathogens have described complement interaction with a variety of bacterial carbohydrate antigens including capsular polysaccharide (Aoyagi Y, Adderson E E, Rubens C E, Bohnsack J F, Min J G, Matsushita M, et al. L-Ficolin/mannose-binding lectin-associated serine protease complexes bind to group B streptococci primarily through N-acetylneuraminic acid of capsular polysaccharide and activate the complement pathway. Infect Immun. 2008 January; 76(1):179-88; Krarup A, Sorensen U B, Matsushita M, Jensenius J C, Thiel S. Effect of capsulation of opportunistic pathogenic bacteria on binding of the pattern recognition molecules mannan-binding lectin, L-ficolin, and H-ficolin. Infect Immun. 2005 February; 73(2):1052-60), peptidoglycan (Nadesalingam J, Dodds A W, Reid K B, Palaniyar N. Mannose-binding lectin recognizes peptidoglycan via the N-acetyl glucosamine moiety, and inhibits ligand-induced proinflammatory effect and promotes chemokine production by macrophages. J Immunol. 2005 Aug. 1; 175(3):1785-94), LTA (Lynch N J, et al. L-ficolin specifically binds to lipoteichoic acid, a cell wall constituent of Gram-positive bacteria, and activates the lectin pathway of complement. J Immunol. 2004 Jan. 15; 172(2):1198-202) and WTA (Park K H, et al. Human serum mannose-binding lectin senses wall teichoic acid Glycopolymer of Staphylococcus aureus , which is restricted in infancy. J Biol Chem. 2010 Aug. 27; 285(35):27167-75).

The profound alterations in accessory cell wall polymers result in an increased susceptibility to complement deposition by the lectin pathway and in increased opsonophagocytic killing of mutant bacteria. The results thus highlight the importance of the structure of accessory cell wall polymers in immune evasion from the complement system.

The present invention will now be further described in the following preferred non-limiting examples with reference to the accompanying figures. For the purposes of the present invention, all references as cited herein are incorporated in their entireties.

FIG. 1 shows opsonophagocytosis and complement deposition of E. faecalis mutants in WTA biosynthesis. A: Opsonophagocytic killing of E. faecalis V583 and WTA biosynthesis mutants in the presence of 1.7% absorbed baby rabbit complement alone, complement in combination with human white blood cells (WBC), or complement, WBC and specific rabbit Ab. Percentage of killing was determined as a relative to colony-forming units from control tubes containing bacteria and WBCs. B: Phagocytosis of FITC-labeled, formalin killed bacteria after 15 min of incubation with human serum. C: C3b deposition on enterococcal bacterial cells measured by FACS after incubation with human serum.

FIG. 2 shows the SDS-PAGE of enzymatically digested cell wall extracts. A Staining with periodic acid and Schiff's reagent (PAS). B Staining with Stains All. Lane 1: molecular mass marker; lane 2: E. faecalis V583 wild type; lane 3: E. faecalis V583Δ1172.

FIG. 3 shows the NMR spectroscopy of two preferred oligosaccharides (OS) I and II of the present invention. A .sup.1H NMR spectra of the anomeric region of OS I and OS II. ROESY spectra of V583 wt OS I (B) and OS II (C) are shown below. Positive signals are depicted in green indicating .sup.1H,.sup.1H connectivities through covalent bonds, negative signals are depicted in blue indicating .sup.1H,.sup.1H ones through the space. All measurements were conducted at 27° C. in D.sub.2O at 700 MHz with the internal standard acetone (δ.sub.H 2.225).

FIG. 4 shows the structures of OS I (A) and OS II (B). EXAMPLES Methods and Materials

Bacterial Strains, Growth Conditions, and Medium

E. faecalis was cultivated at 37° C. without agitation in tryptic soy broth (TSB) (Carl Roth, Karlsruhe) which was supplemented with the respective antibiotics as indicated. Short term cultures were grown on tryptic soy agar (TSA) and stored at 4° C. Cultures for isolation of enterococcal cell wall polysaccharides were grown to stationary phase for 18 h and harvested by centrifugation. E. coli cultures were cultivated aerobically in Luria Bertani (LB) medium (Carl Roth, Karlsruhe) which was supplemented with antibiotics as indicated.

Construction of an EF1172 Gene Insertion Mutant in E. faecalis V583

A targeted insertion mutant of gene EF_1172 in E. faecalis V583 was done as described by Rigottier-Gois et al. (Rigottier-Gois L, Alberti A, Houel A, Taly J-F, Palcy P, Manson J, et al. Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors. PLoS ONE. 2011 Dec. 15; 6(12):e29023), and the localization of the targeted insertion was verified by PCR and Southern blot as described previously.

Sera and Abs in Complement Binding Studies

Normal pooled human serum was obtained from healthy volunteers, who gave informed consent. Baby rabbit serum was obtained from Cederlane Laboratories. Clq-depleted serum was purchased from Quidel. Heat-inactivation of serum was performed by incubation for 20 min at 56° C. Serum was depleted of specific Ab to E. faecalis V583 by absorption with bacterial cells at 4° C. for 60 min. After absorption, bacteria were pelleted by centrifugation and the supernatant was passed through a 0.2 μm filter.

Opsonophagocytic Killing Assay

Bacterial opsonophagocytosis by human white blood cells (WBCs) was measured as described previously (Theilacker C, et al. Serodiversity of Opsonic Antibodies against Enterococcus faecalis -Glycans of the Cell Wall Revisited. PLoS ONE. 2011; 6(3):e17839). Briefly, bacterial strains were grown to mid-logarithmic phase (OD.sub.600=0.4) in TSB and diluted with RPMI supplemented with 15% fetal calf serum. WBCs were purified from the blood of healthy volunteers by sedimentation with heparin-dextran buffer and remaining erythrocytes were removed by hypotonic lysis in 1% NH.sub.4Cl solution. Baby rabbit serum diluted 1:15 served as complement source. To remove natural Abs against target strains the complement was absorbed with E. faecalis V583 as described above. Rabbit serum raised against heat-killed E. faecalis V583 at a dilution of 1:2500 served as Ab source. In control tubes, Ab, complement, or PMNs were omitted from the assay. For the measurement of opsonophagocytosis, equal volumes of 2.5×10.sup.6 PMNs, 2.5×10.sup.6 CFUs bacteria, complement and heat-inactivated immune rabbit serum were combined. After 90 min incubation, the reaction was stopped at 4° C., PMNs were lyzed by hypotonic lysis, and viable cell counts were determined by plating of serial dilutions.

Phagocytosis Assay

The phagocytosis assay was performed as described with modifications (Rooijakkers S H, et al. Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases. Nat Immunol. 2005 September; 6(9):920-7). Human pooled serum was absorbed with the E. faecalis as described above and diluted in RPMI with addition of 0.05% human serum albumin (HSA) to the respective concentration. Next, 7.5×10.sup.4 freshly isolated human neutrophils and 7.5×10.sup.5 FITC-labeled, heat-killed E. faecalis V583 wild type or E. faecalis V583Δ1172 were added and incubated for 15 min at 37° C. while shaking at 750 rpm. The reaction was stopped by adding 1.5% ice-cold paraformaldehyde in RPMI 0.1% HSA. Phagocytosis was analyzed by flow cytometry (FACSCalibur; Becton Dickinson) measuring fluorescence of 10,000 gated neutrophils. Heat-inactivated serum was used as a control for complement-independent phagocytosis.

C3b Deposition on E. faecalis

E. faecalis strains were grown to an OD.sub.600 of 0.5 in TSB and washed in HEPES.sup.++ buffer (20 mM HEPES, 140 mM NaCl, 5 mM CaCl.sub.2, 2.5 mM MgCl.sub.2) with 0.1% BSA. 12.5×10.sup.5 bacteria were incubated with serum for 30 min while shaking at 900 rpm. Bacteria were washed with PBS supplemented with 0.1% BSA. C3b deposition was detected using mouse anti-human C3d Abs (Quidel) and FITC-conjugated goat anti-mouse IgG (Protos). Fluorescence of 10,000 bacteria was measured by flow cytometry. Classical and lectin pathway was abolished by incubation bacteria with complement in the presence HEPES.sup.++ buffer plus 5 mM MgCl.sub.2 and 10 mM EGTA, pH 7.5. For specific inactivation of the classical pathway, Clq depleted serum was used (Quidel). The C3 convertase complex (C4bC2a) was measured using a monoclonal antibody to human C4/C4d (Quidel) and goat polyclonal anti-mouse FITC conjugate as secondary antibody (Dako). MASP-2 deposition was detected using a goat polyclonal anti-MASP-2 Ab (Santa Cruz Biotechnology).

Isolation of Cell-Wall Polysaccharide

For isolation of enterococcal cell wall polysaccharides, bacteria were cultivated as described above. Bacteria were washed and resuspended in digestion buffer (PBS plus 20 mM CaCl.sub.2, 20 mM MgCl.sub.2, NaN.sub.3 0.05%) and cleaved enzymatically with mutanolysin (0.01 mg/mL) and lysozyme (0.5 mg/mL) at 37° C. for 18 h. Afterwards, insoluble material was removed by centrifugation and the supernatant was treated with nucleases (DNase I and RNase, final concentration 0.1 mg/mL) for 18 h at 37° C. Proteins were degraded by digestion with proteinase K (0.1 mg/mL) for 8 h at 56° C. The supernatant was extensively dialyzed (10 ku MMCO) against H.sub.2O and lyophilized. For size exclusion chromatography (SEC), the sample was dissolved in 50 mM ammoniumcarbonate buffer (pH 8.8; NaN.sub.3 0.02%) and applied on a Sephacryl S 200 column (1.6×100 cm, GE Healthcare). Hexose and phosphorus content were measured as described previously (Theilacker C, et al. Opsonic antibodies to Enterococcus faecalis strain 12030 are directed against lipoteichoic acid. Infect Immun. 2006 October; 74(10):5703-12), and positive fractions eluting at a K.sub.av of 0.29 and 0.31, respectively, were combined, dialyzed and lyophilized. The resulting material was dissolved in 20 mM NaHCO.sub.3 (pH 8.0, NaN.sub.3 0.02%) and subjected to anion-exchange chromatography (Q Sepharose FF, GE Healthcare).

To cleave phosphodiester bonds, 10 mg sample was dissolved in 50 μL 48% HF and incubated at 4° C. for 2 d. The material was neutralized and separated by SEC on Sephadex G50 (1.6×100 cm column, Biorad). Fractions of the lowest molecular mass were further purified by SEC on Biogel P2 (1×120 cm Column, Biorad), followed by high-performance anion-exchange chromatography (HPAEC, Dionex) applying a CarboPak PA 100 column (9×250 mm) and an ED50 electrochemical detector (Dionex). Data analysis was performed using the Chromeleon Version 6.6 software.

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